There is a continuing need in medical, dental and veterinary practices, in research and diagnostic procedures, and for rapid and accurate detection or quantification of biological or chemical substances present in biological fluids or specimens. For example, the presence of various microorganisms in human and animal tissues, fluids or cells is very important for diagnosis and effective treatment of diseases.
Specific microorganisms have been implicated as markers for a number of periodontal diseases in humans and animals, such as gingivitis and periodontitis. The importance of such diseases is growing in the human population, especially as people live longer, and prevention thereof is becoming of considerable medical and commercial importance. In addition, the proper care of animals (including dental care) is a growing concern in our culture.
Detection of microorganisms associated with periodontal diseases has been accomplished using culture techniques (which are generally slow), DNA probes and a number of immunological procedures, such as agglutination assays, enzyme linked immunosorbent assays (ELISA), immunofluorescence and immunoprecipitation. The immunological procedures use immunological reactions of an antigenic site of the microorganism (which may be on a component extracted therefrom) with a corresponding antibody specific thereto. The resulting immunological reaction complex can then be detected in a number of ways.
The black-pigmented Bacteroides species are gram negative, anaerobic rods which are common in the etiology of various periodontal diseases, odontogenic abscesses and endodontic lesions. Such species include Bacteroides intermedius, Bacteroides(Porphyromonas) gingivalis, Bacteroides forsythus and Bacteroides endodontalis. Bacteroides gingivalis was recently renamed as Porphyromonas gingivalis. As defined herein, periodontal disease refers to a wide variety of diseases in humans and animals which occur in the periodontal area of the oral cavity, and includes diseases affecting the cemental surfaces of the roots of teeth, alveolar bone and the gingiva. It includes diseases that may occur at different times of human life, such as in infancy, youth, pregnancy or old age.
It is also known that certain serogroups or serotypes within a bacterial species which causes periodontal disease are responsible for certain forms of those diseases. For example, it is believed by some [Nakazawa et al, Infect. Immun., 56(6), pp. 1647-1651, 1988] that serotype B of Bacteroides intermedius is responsible for human adolescent periodontitis, while serotypes B and C are responsible for adult periodontitis.
Clinical assays specific to such bacteria in gingival crevicular fluid and subgingival dental plaque are useful in the diagnosis of periodontal disease, in evaluating the options for and the progress of periodontal treatment, and in determining the status of the patient at later dental examinations. The standard culture techniques used to identify such organisms are time consuming, expensive and require a high level of operator expertise. Such tests also may lack sufficient sensitivity for detection of low levels of organisms due to strict anaerobic conditions required during transport.
The various immunoassays noted above have been developed and used with some success. Particularly useful are radioimmunoprecipitation assays and ELISA tests. U.S. Pat. No. 4,741,999 (issued May 3, 1988 to Genco et al) describes the production of monoclonal antibodies to antigens of Actinobacillus actinomycetemcomitans and their use in ELISA assays.
Monoclonal and polyclonal antibodies to various Bacteroides species have been prepared for similar assays [see for example Nakazawa et al, noted above and Zambon et al, J. Periodon., 56(Supp), pp. 32-40, 1985] .
Generally, the assays of the art have been slow, tedious and directed to a single organism. In many cases, the assays exhibit considerable cross-reactivity with related species, and thus have limited usefulness.
In copending U.S. Ser. No. 468,034 (filed on even date herewith by Snyder and entitled "Screening Assay for Microorganisms Associated with Periodontal Diseases, Article and Kit Useful Therein", now abandoned), a screening assay is described for detecting the presence of any of the three microorganisms Actinobacillus actinomycetemcomitans, Bacteroides(Porphyromonas) gingivalis and Bacteroides intermedius. This screening method is highly useful for practitioners so they can initially determine if a patient needs periodontal treatment.
However, once screening has been done, and the results are found to be positive, it would be desirable to be able to determine which microorganisms are present so the treatment can be prescribed specifically. Such treatment could then begin early. Presently, differentiation is carried out using the tedious culture or undesirable immunological methods described above.
Moreover, it would be useful to have an assay which is much more rapid, but as sensitive as known culture techniques.